Development and maintenance of immune tolerance is critical to minimizing complications of allogeneic hematopoietic cell transplantation (HCT) for non-malignant hematologic disorders, allowing donor cell engraftment while minimizing graft-versus-host disease (GVHD). Harnessing innate immune mechanisms has great potential to maintain immune tolerance across histocompatibility barriers. However, the mechanisms through which innate immune regulation of the adaptive immune system can be achieved after allogeneic HCT are poorly understood. Understanding these mechanisms will allow us to better apply them to generate immune tolerance between transplant donor and recipient, thus expanding alternative donor HCT to cure non-malignant blood disorders. We recently described novel innate mechanisms through which recipient regulatory myeloid dendritic cells (MDC) spared by non-marrow ablative total lymphoid irradiation (TLI) and T cell- depletive anti-thymocyte serum (ATS) can induce the proliferation of Foxp3+ regulatory T cells in the donor graft across major histocompatibility complex (MHC) barriers. We have since determined a modified pre-transplant preparative regimen which augments the recovery of these regulatory myeloid cells and key signaling mechanisms required for regulatory function, and which results in durable donor-recipient immune tolerance after MHC-mismatched HCT in thalassemic mice. Our central hypotheses are that a group of recipient myeloid precursor cells spared by non-myeloablative conditioning develop into regulatory MDC through direct interactions with another recipient innate immune cell population, and that these myeloid precursors thus play a central role in regulating donor immune responses after MHC-mismatched HCT. We will test these hypotheses through 4 specific questions: 1. Can these cells regulate graft-versus-host immune responses after HCT? 2. How are these myeloid cells formed under the influence of other specific innate immune cells of the recipient? 3. What specific signaling mechanisms drive recipient myeloid cell-induced immune tolerance after this conditioning? 4. Can these myeloid regulatory cells be generated when combining other drugs of the same class with the TLI/ATS regimen, and can one of these regimens be optimized for sickle cell disease (SCD) using high-fidelity murine models of human SCD? Our studies should provide critical insights into specific innate mechanisms of regulation of MHC-mismatched transplantation tolerance and new therapeutic options in HCT for hemoglobinopathies, health conditions with high global prevalence and relevance.